Downregulation of the CCL2/CCR2 and CXCL10/CXCR3 axes contributes to antitumor effects in a mouse model of malignant glioma

Glioblastoma multiforme involves glioma stem cells (GSCs) that are resistant to various therapeutic approaches. Here, we studied the importance of paracrine signaling in the glioma microenvironment by focusing on the celecoxib-mediated role of chemokines C–C motif ligand 2 (CCL2), C-X-C ligand 10 (CXCL10), and their receptors, CCR2 and CXCR3, in GSCs and a GSC-bearing malignant glioma model. C57BL/6 mice were injected with orthotopic GSCs intracranially and divided into groups administered either 10 or 30 mg/kg celecoxib, or saline to examine the antitumor effects associated with chemokine expression. In GSCs, we analyzed cell viability and expression of chemokines and their receptors in the presence/absence of celecoxib. In the malignant glioma model, celecoxib exhibited antitumor effects in a dose dependent manner and decreased protein and mRNA levels of Ccl2 and CxcL10 and Cxcr3 but not of Ccr2. CCL2 and CXCL10 co-localized with Nestin+ stem cells, CD16+ or CD163+ macrophages and Iba-1+ microglia. In GSCs, celecoxib inhibited Ccl2 and Cxcr3 expression in a nuclear factor-kappa B-dependent manner but not Ccr2 and CxcL10. Moreover, Ccl2 silencing resulted in decreased GSC viability. These results suggest that celecoxib-mediated regulation of the CCL2/CCR2 and CXCL10/ CXCR3 axes may partially contribute to glioma-specific antitumor effects

偏光分解顕微鏡を用いたアルコール性肝線維症の線維化の量的・質的評価系の開発

Liver fibrosis is assessed mainly by conventional staining or second harmonic generation (SHG) microscopy, which can only provide collagen content in fibrotic area. We propose to use polarization-resolved SHG (PR-SHG) microscopy to quantify liver fibrosis in terms of collagen fiber orientation and crystallization. Liver samples obtained from autopsy cases with fibrosis stage of F0–F4 were evaluated with an SHG microscope, and 12 consecutive PR-SHG images were acquired while changing the polarization azimuth angle of the irradiated laser from 0° to 165° in 15° increments using polarizer. The fiber orientation angle (φ) and degree (ρ) of collagen were estimated from the images. The SHG-positive area increased as the fibrosis stage progressed, which was well consistent with Sirius Red staining. The value of φ was random regardless of fibrosis stage. The mean value of ρ (ρ-mean), which represents collagen fiber crystallinity, varied more as fibrosis progressed to stage F3, and converged to a significantly higher value in F4 than in other stages. Spatial dispersion of ρ (ρ-entropy) also showed increased variation in the stage F3 and decreased variation in the stage F4. It was shown that PR-SHG could provide new information on the properties of collagen fibers in human liver fibrosis

A reduced brain and liver FDG uptake

Purpose : To investigate whether or not the physiological brain and liver FDG uptake are decreased in patients with highly accelerated glycolysis lesions. Methods : We retrospectively analyzed 51 patients with malignant lymphoma. We compared the FDG uptake in the brain and liver of the patients with that in a control group. In 24 patients with a complete response (CR) or partial response (PR) to treatment, we compared the brain and liver uptake before and after treatment. Results : The maximum standardized uptake value (SUVmax) and total glycolytic volume (TGV) of the brain as well as the SUVmax and mean standardized uptake value (SUVmean) of the liver in malignant lymphoma patients were 13.1 ± 2.3, 7386.3 ± 1918.4, 3.2 ± 0.5, and 2.3 ± 0.4, respectively ; in the control group, these values were 14.9 ± 2.4, 8566.2 ± 1659.5, 3.4 ± 0.4, and 2.5 ± 0.3, respectively. The SUVmax and TGV of the brain and the SUVmean of the liver in malignant lymphoma patients were significantly lower than the control group. The SUVmax and TGV of the brain after treatment were significantly higher than before treatment. Both the SUVmax and SUVmean of liver after treatment were higher than before treatment, but not significant. Conclusion : A decreased physiological brain and liver FDG uptake is caused by highly accelerated lesion glycolysis

Functional analysis of HOXD9 in human gliomas and glioma cancer stem cells

<p>Abstract</p> <p>Background</p> <p><it>HOX </it>genes encode a family of homeodomain-containing transcription factors involved in the determination of cell fate and identity during embryonic development. They also behave as oncogenes in some malignancies.</p> <p>Results</p> <p>In this study, we found high expression of the <it>HOXD9 </it>gene transcript in glioma cell lines and human glioma tissues by quantitative real-time PCR. Using immunohistochemistry, we observed HOXD9 protein expression in human brain tumor tissues, including astrocytomas and glioblastomas. To investigate the role of <it>HOXD9 </it>in gliomas, we silenced its expression in the glioma cell line U87 using <it>HOXD9</it>-specific siRNA, and observed decreased cell proliferation, cell cycle arrest, and induction of apoptosis. It was suggested that <it>HOXD9 </it>contributes to both cell proliferation and/or cell survival. The <it>HOXD9 </it>gene was highly expressed in a side population (SP) of SK-MG-1 cells that was previously identified as an enriched-cell fraction of glioma cancer stem-like cells. <it>HOXD9 </it>siRNA treatment of SK-MG-1 SP cells resulted in reduced cell proliferation. Finally, we cultured human glioma cancer stem cells (GCSCs) from patient specimens found with high expression of <it>HOXD9 </it>in GCSCs compared with normal astrocyte cells and neural stem/progenitor cells (NSPCs).</p> <p>Conclusions</p> <p>Our results suggest that <it>HOXD9 </it>may be a novel marker of GCSCs and cell proliferation and/or survival factor in gliomas and glioma cancer stem-like cells, and a potential therapeutic target.</p

Blocking COX-2 induces apoptosis and inhibits cell proliferation via the Akt/survivin- and Akt/ID3 pathway in low-grade-glioma

Approximately half of surgically-treated patients with low-grade-glioma (LGG) suffer recurrence or metastasis. Currently there is no effective drug treatment. While the selective COX-2 inhibitor celecoxib showed anti-neoplastic activity against several malignant tumors, its effects against LGG remain to be elucidated. Ours is the first report that the expression level of COX-2 in brain tissue samples from patients with LGG and in LGG cell lines is higher than in the non-neoplastic region and in normal brain cells. We found that celecoxib attenuated LGG cell proliferation in a dose-dependent manner. It inhibited the generation of prostaglandin E2 and induced apoptosis and cell-cycle arrest. We also show that celecoxib hampered the activation of the Akt/survivin- and the Akt/ID3 pathway in LGGs. These findings suggest that celecoxib may have a promising therapeutic potential and that the early treatment of LGG patients with the drug may be beneficial

Tumour resistance in induced pluripotent stem cells derived from naked mole-rats

The naked mole-rat (NMR, Heterocephalus glaber), which is the longest-lived rodent species, exhibits extraordinary resistance to cancer. Here we report that NMR somatic cells exhibit a unique tumour-suppressor response to reprogramming induction. In this study, we generate NMR-induced pluripotent stem cells (NMR-iPSCs) and find that NMR-iPSCs do not exhibit teratoma-forming tumorigenicity due to the species-specific activation of tumour-suppressor alternative reading frame (ARF) and a disruption mutation of the oncogene ES cell-expressed Ras (ERAS). The forced expression of Arf in mouse iPSCs markedly reduces tumorigenicity. Furthermore, we identify an NMR-specific tumour-suppression phenotype—ARF suppression-induced senescence (ASIS)—that may protect iPSCs and somatic cells from ARF suppression and, as a consequence, tumorigenicity. Thus, NMR-specific ARF regulation and the disruption of ERAS regulate tumour resistance in NMR-iPSCs. Our findings obtained from studies of NMR-iPSCs provide new insight into the mechanisms of tumorigenicity in iPSCs and cancer resistance in the NMR

RNA-Binding Protein Musashi1 Modulates Glioma Cell Growth through the Post-Transcriptional Regulation of Notch and PI3 Kinase/Akt Signaling Pathways

Musashi1 (MSI1) is an RNA-binding protein that plays critical roles in nervous-system development and stem-cell self-renewal. Here, we examined its role in the progression of glioma. Short hairpin RNA (shRNA)-based MSI1-knock down (KD) in glioblastoma and medulloblastoma cells resulted in a significantly lower number of self renewing colony on day 30 (a 65% reduction), compared with non-silencing shRNA-treated control cells, indicative of an inhibitory effect of MSI1-KD on tumor cell growth and survival. Immunocytochemical staining of the MSI1-KD glioblastoma cells indicated that they ectopically expressed metaphase markers. In addition, a 2.2-fold increase in the number of MSI1-KD cells in the G2/M phase was observed. Thus, MSI1-KD caused the prolongation of mitosis and reduced the cell survival, although the expression of activated Caspase-3 was unaltered. We further showed that MSI1-KD glioblastoma cells xenografted into the brains of NOD/SCID mice formed tumors that were 96.6% smaller, as measured by a bioluminescence imaging system (BLI), than non-KD cells, and the host survival was longer (49.3±6.1 days vs. 33.6±3.6 days; P<0.01). These findings and other cell biological analyses suggested that the reduction of MSI1 in glioma cells prolonged the cell cycle by inducing the accumulation of Cyclin B1. Furthermore, MSI1-KD reduced the activities of the Notch and PI3 kinase-Akt signaling pathways, through the up-regulation of Numb and PTEN, respectively. Exposure of glioma cells to chemical inhibitors of these pathways reduced the number of spheres and living cells, as did MSI1-KD. These results suggest that MSI1 increases the growth and/or survival of certain types of glioma cells by promoting the activation of both Notch and PI3 kinase/Akt signaling

Effects of rapamycin on <i>pten</i> dko embryos.

<p>(<b>A</b>) A total of 41 embryos obtained from <i>ptena</i>^+/−<i>ptenb</i>^−/− parents was treated with 5 μM rapamycin for 96 h beginning at 26 hpf. (<b>B</b>) At 7 dpf, the embryos were photographed and genotyped. Eight of the 41 embryos had the <i>ptena</i>^−/−<i>ptenb</i>^−/− genotype (images 1–8), nine were <i>ptena</i>^+/−<i>ptenb</i>^−/−(image 9), and 10 were <i>ptena</i>^+/+<i>ptenb</i>^−/−(image 10). Partial Cuvierian ducts developed in seven of the eight dko embryos. X, no duct. (<b>C</b>) Snapshots from movies of the dko embryos shown in images 2 (upper) and 4 (lower) in (B). Blood cells can be seen flowing through the Cuvierian duct (upper) and the tail vascular duct (lower) indicated by the arrows. Times are in seconds.</p

Establishment of <i>pten</i> knockout medaka with transcription activator–like effector nucleases (TALENs) as a model of PTEN deficiency disease

<div><p>Phosphatase and tensin homolog (PTEN) is a lipid and protein phosphatase that antagonizes signaling by the phosphatidylinositol 3-kinase (PI3K)–AKT signaling pathway. The <i>PTEN</i> gene is a major tumor suppressor, with mutations of this gene occurring frequently in tumors of humans and mice. We have now developed mutant medaka deficient in PTEN with the use of transcription activator–like effector nuclease (TALEN) technology. Medaka possesses two <i>pten</i> genes, <i>ptena</i> and <i>ptenb</i>, similar to zebrafish. We established 16 <i>ptena</i> mutant lines and two <i>ptenb</i> mutant lines. Homozygous single <i>pten</i> mutants were found to be viable and fertile. In contrast, <i>pten</i> double-knockout (dko) embryos manifested severe abnormalities in vasculogenesis, eye size, and tail development at 72 hours post fertilization(hpf) and died before hatching. Immunoblot analysis revealed that the ratio of phosphorylated to total forms of AKT (pAKT/AKT) in <i>pten</i> dko embryos was four times that in wild-type embryos, indicative of up-regulation of signaling by the PI3K-AKT pathway. Treatment of <i>pten</i> dko embryos with the PI3K inhibitor LY294002 reduced the pAKT/AKT ratio by about one-half and partially rescued the defect in vasculogenesis. Additional inhibitors of the PI3K-AKT pathway, including rapamycin and <i>N</i>-α-tosyl-L-phenylalanyl chloromethyl ketone, also partially restored vasculogenesis in the dko embryos. Our model system thus allows <i>pten</i> dko embryos to be readily distinguished from wild-type embryos at an early stage of development and is suitable for the screening of drugs able to compensate for PTEN deficiency.</p></div